CN105002270A - Primers and probes for specifically detecting enterobacteriaceae bacteria and use thereof - Google Patents
Primers and probes for specifically detecting enterobacteriaceae bacteria and use thereof Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention belongs to the technical field of microbiological detection, relates to primers and probes for specifically detecting enterobacteriaceae bacteria and a use thereof, and further discloses a method for specifically detecting enterobacteriaceae bacteria. The primers and probes for specifically detecting enterobacteriaceae bacteria and synchronously detecting enterobacteriaceae bacteria and enterobacter sakazakii are specifically designed according to bacterial strain characteristics so that result accuracy is ensured.
Description
Technical field
The invention belongs to technical field of microbial detection, be specifically related to a kind of primer of specific detection enterobacteriaceae lactobacteriaceae, probe and application thereof, and further disclose a kind of method of specific detection enterobacteriaceae lactobacteriaceae.
Background technology
Enterobacteriaceae lactobacteriaceae is under the jurisdiction of bacterium circle, enterobacteriaceae comprises without gemma, whole body flagellum or atrichous Gram-negative straight-bar bacterium, there is widespread feature, its host range is large, people, animal, plant have parasitism or symbiosis, grow nonparasitically upon another plant, saprophytic, also can survive in soil or water, close with human relation.Enterobacteriaceae lactobacteriaceae is easy to cultivate, breeding fast (can breed a generation in every 20 ~ 30 minutes under optimum conditions).
Enterobacteriaceae lactobacteriaceae has chemoorganotrophy, runs respiratory metabolism and fermentating metabolism concurrently; By being oxidized multiple simple organic compound or sugar fermentation, organic acid or polyvalent alcohol to obtain energy; Major part can grow on the inorganic nitrogen substratum containing a kind of carbon source; Some needs certain seed amino acid or water-soluble vitamins when planting growth; Except individual serotype, catalase is the positive, oxidase negative; Except the minority kind in erwinia, the nitrate that all reduces is nitrite; G+C (guanine and cytosine(Cyt)) mol content in DNA (thymus nucleic acid) is 39 ~ 59%.
And Enterobacter sakazakii, belong to enterobacteriaceae, colonize in the Gram-negative bacteria in humans and animals enteron aisle, without gemma, peritrichous, can move, it comprises the thalline that Escherichia, salmonella, Shigella, Klebsiella, serratia, proteus, Yersinia etc. belong to.Enterobacter sakazakii can cause baby and premature infant's meningitis, septicemia and necrotic colitis, and mortality ratio, up to 50%, has caused the attention of multinational relevant departments of the world.Country's food origin disease monitoring net was classified as Enterobacter sakazakii as infant formula emphasis monitoring pathogenic bacteria from 2007.
Current most of Chinese Enterprise adopts the Enterobacter sakazakii in Screening of Media method detection infant formula, and concrete operations are with reference to GB 478940-2010, and operation steps is as follows:
(1) front increasing bacterium and increasing bacterium
Get sample 100g (mL) to add and be preheated to 44 DEG C and be equipped with in the Erlenmeyer flask of 900mL buffered peptone water, gently shake to abundant dissolving with hand, cultivate 18h ± 2h for 36 DEG C ± 1 DEG C.Pipette 1mL transferred species in 10mL mLST-Vm meat soup, cultivate 24h ± 2h for 44 DEG C ± 0.5 DEG C.
(2) be separated
2.1 mix mLST-Vm broth culture gently, respectively get Zengjing Granule thing 1 ring, and streak inoculation is in two Enterobacter sakazaii colour development culture medium flat boards respectively, cultivate 24h ± 2h for 36 DEG C ± 1 DEG C.
2.2 pickings 1 ~ 5 suspicious bacterium colonies, streak inoculation is dull and stereotyped in tryptose soya agar (TSA).Cultivate 48h ± 4h for 25 DEG C ± 1 DEG C.
(3) identify
On TSA flat board, the yellow suspicious bacterium colony of direct picking, carries out biochemical identification.Biochemical identification test kit or full automatic microorganism biochemical identification system can be selected.
The shortcoming of above-mentioned Screening of Media method is as follows:
1, total detection time needs about 5 days, and the culture dish needing inoculation different in a large number, waste time and energy.
2, mass propgation base starting material are needed, consumptive material etc.
3, need a large amount of spaces to place these culture dish and need a large amount of manpower cleaning culture dish.
The detection technique (People's Republic of China (PRC) inspection and quarantining for import/export industry standard SN/T1632.3-2005) of fluorescence PCR method is also had except Screening of Media method.PCR full name is polymerase chain reaction.Using two sections of (20-24 Nucleotide) oligonucleotide as the primer of reaction, there is not complementary action in the sequence of these two sections of Oligonucleolide primers.But complementation can be there is respectively with the site become on DNA to be measured two chains of template in them.Reaction solution is by the reaction buffer comprised containing magnesium ion, and archaeal dna polymerase, 4 kinds of mononucleotides (dNTP), template DNA and primer form.The DNA fragmentation between two complementary sites is synthesized by the change (sex change, annealing extend) of temperature.Such reaction is carried out repeatedly, and DNA fragmentation is increased.Through about 30 circulations, amplification times reaches 106.
And fluorescent PCR adds a special oligonucleotide fluorescent probe on regular-PCR basis, this probe 5 ' end marked FAM fluorescein, and 3 ' end marked TAMRA fluorescein, and now the fluorescence of FAM is by TAMRA quencher, and instrument can't detect fluorescence.PCR extends the stage, archaeal dna polymerase play its 5 '-3 ' exonuclease function, cut by probe, be meanwhile marked at FAM on probe free out, its fluorescence sent no longer is detected by instrument by TAMRA institute's quencher.In PCR process, often pair of object fragment once effectively increases, and has carried out once cutting and having carried out one-time detection to FAM to probe simultaneously.Instrument detects the increment of FAM, indirectly can reflect the amplification amount of object fragment.Concrete operation step is as follows:
(1) sample, got after 1mL increasing bacterium is added in 1.5mL sterile centrifugation tube;
(2), the centrifugal 5min of 8000r/min, remove supernatant liquor as far as possible;
(3), add 50 μ LDNA extracting solutions, fully mix;
(4), boiling water bath 5min;
(5), the centrifugal 5min of 12000r/min.Namely supernatant liquor can be used as template DNA and carries out fluorescent PCR amplification;
(6), reaction system cumulative volume is 25 μ L.Wherein contain: 10 times of damping fluid 2.5 μ L, primer pair (10 μm of ol/L) each 1 μ L, dNTP (10mmol/L) 1 μ L, archaeal dna polymerase (5U/ μ L) 0.5 μ L, water 17 μ L, template DNA 2 μ L;
(7), response procedures is: 1.37 DEG C of 5min, 95 DEG C of denaturation 3min; 2.95 DEG C of sex change 5s, 60 DEG C of annealing extend 40s, 40 circulations, collect FAM fluorescence simultaneously;
(8), again, hybridization needs to be equipped with special hybrid heater, takes up room many, and control condition is complicated, is difficult to realize high-throughput.
The shortcoming of above-mentioned fluorescent PCR method detection technique is as follows:
1, specificity is lower: due to the problem of primer and probe design, for the detection of food conditional pathogenic bacterium, almost only be confined to check a kind of pathogenic bacterium, inspection while seldom realizing a step experiment, can not comprise all bacterial classifications of Enterobacter sakazakii and enterobacteria completely.
2, false positive rate is high: dead Enterobacter sakazakii is harmless, but still can detect.Accumulation due to dead cell shows the high background of the coli cell of inactivation, and the aerosol of DNA fragmentation formation free in experimental implementation process all can cause the false positive of experimental result, thus can increase workload on foot because continuing subsequent experimental.
3, false negative rate is high: can not get rid of the false negative phenomenon occurred due to the existence of PCR inhibitor.
4, the more difficult judgement of result: simple with Ct value result of determination, accurately rigorous not.
Visible, the detection method of existing routine all has its drawback for the detection of enterobacteriaceae lactobacteriaceae especially Enterobacter sakazakii, and needing that exploitation is a kind of badly can the method for specific detection enterobacteriaceae lactobacteriaceae especially Enterobacter sakazakii fast and accurately.
Summary of the invention
Technical problem to be solved by this invention be to provide a kind of can the primer of specific detection enterobacteriaceae lactobacteriaceae, probe and application thereof, and further disclose a kind of method of specific detection enterobacteriaceae lactobacteriaceae;
Second technical problem that the present invention solves be to provide a kind of can detect enterobacteriaceae lactobacteriaceae and Enterobacter sakazakii simultaneously primer, probe and application thereof, and further disclose a kind of method simultaneously detecting enterobacteriaceae lactobacteriaceae and Enterobacter sakazakii.
In order to solve the problem, the invention provides a kind of primer of specific detection enterobacteriaceae lactobacteriaceae, the sequential structure of described primer is as follows:
Enterobacteriaceae-F:5 '-CTG AAA GCA TCT AAG CAC GAA ACT TG-3 ';
Enterobacteriaceae-R:5 '-GTC GTC TTC AAC GTT CCT TCA GG-3 ';
Internal positive control-F:5 '-TGT GAA ATA CCG CAC AGA TG-3 ';
Internal positive control (IAC)-R:5 '-AGC TGG CGT AAT AGC GAA G-3 '.
Present invention also offers a kind of probe for specific detection Enterobacter sakazakii, the sequential structure of described probe is as follows:
Enterobacteriaceae lactobacteriaceae probe:
5′-HEX-AGAGTAGTAGTTGTAGAGGCCGTGCTTCCGAAAG-BHQ-1-3′；
Internal positive control probe:
5′-ROX-TAAGGAGAAAATACCGCATCAGGCGCC-BHQ-2-3′。
Present invention also offers a kind of test kit for specific detection enterobacteriaceae lactobacteriaceae, comprise described primer and described probe, and sensitivity of light material.
Described sensitivity of light material comprises and can form ethidium bromide list nitrine bromine (EMA) of covalent linkage with the base of DNA, and its concentration is 125 μ g/mL.
Further, described test kit also comprises:
Internal positive control thing, described internal positive control thing comprises pUC19 plasmid DNA;
The mixture of archaeal dna polymerase and uracil-DNA glycosylase;
The DNA of enterobacteriaceae lactobacteriaceae is positive control, the contrast of PCR level water belongs with yin;
Buffered peptone water and DNA extraction liquid;
And optionally add visual yellow dyes.
The invention also discloses a kind of method of specific detection enterobacteriaceae lactobacteriaceae, namely adopt described test kit, this detection method comprises the following steps:
(1) extraction of DNA:
Get measuring samples and carry out multiplication culture, enrichment, 300 μ l ethidium bromide list nitrine bromines are added in 100 μ l pregnant solutions, then 5min is cultivated in room temperature darkroom, 5min is exposed afterwards under 500W light, centrifugal, removing supernatant liquor, add DNA extraction liquid again, fully mixing, cultivate, centrifugal, get supernatant liquor as template DNA;
(2) PCR reaction system:
The reaction system of 25 μ L comprises primer and probe described in 18 μ L; The described archaeal dna polymerase of 1 μ L and the mixture of uracil-DNA glycosylase; 1 μ L internal positive control thing; 5 μ L template DNAs or positive control or negative control thing;
(3) PCR response procedures:
4min at 37 DEG C, 95 DEG C of denaturation 5min; 95 DEG C of sex change 10s, 65 DEG C of annealing extend 70s, and 40 circulations, collect FAM, HEX and ROX fluorescent signal simultaneously;
(4) experimental result is judged in conjunction with amplification curve diagram.
Present invention also offers a kind of test kit for specific detection enterobacteriaceae and Enterobacter sakazakii, comprising:
Enterobacter sakazakii-F:5 '-AGG GGA TAT TGT CCC CTG AAA CAG-3 ';
Enterobacter sakazakii-R:5 '-AAA CGA GAA TAA GCC GCG CAT T-3 ';
Enterobacteriaceae-F:5 '-CTG AAA GCA TCT AAG CAC GAA ACT TG-3 ';
Enterobacteriaceae-R:5 '-GTC GTC TTC AAC GTT CCT TCA GG-3 ';
Internal positive control-F:5 '-TGT GAA ATA CCG CAC AGA TG-3 ';
Internal positive control (IAC)-R:5 '-AGC TGG CGT AAT AGC GAA G-3 ';
Enterobacter sakazakii probe:
5′-HEX-AGAGTAGTAGTTGTAGAGGCCGTGCTTCCGAAAG-BHQ-1-3′；
Enterobacteriaceae probe:
5′-HEX-AGAGTAGTAGTTGTAGAGGCCGTGCTTCCGAAAG-BHQ-1-3′；
Internal positive control probe:
5′-ROX-TAAGGAGAAAATACCGCATCAGGCGCC-BHQ-2-3′；
And sensitivity of light material.
Present invention also offers a kind of test kit for specific detection enterobacteriaceae and Enterobacter sakazakii, described sensitivity of light material comprises and can form ethidium bromide list nitrine bromine (EMA) of covalent linkage with the base of DNA, and its concentration is 125 μ g/mL.
Described test kit also comprises:
Internal positive control thing, described internal positive control thing comprises pUC19 plasmid DNA;
The mixture of archaeal dna polymerase and uracil-DNA glycosylase;
The DNA of enterobacteriaceae lactobacteriaceae is positive control, the contrast of PCR level water belongs with yin;
Buffered peptone water and DNA extraction liquid;
And optionally add visual yellow dyes.
Present invention also offers a kind of method of specific detection enterobacteriaceae lactobacteriaceae and Enterobacter sakazakii, it is characterized in that, the test kit described in employing, this detection method comprises the following steps:
(1) extraction of DNA:
Get measuring samples and carry out multiplication culture, enrichment, 300 μ l ethidium bromide list nitrine bromines are added in 100 μ l pregnant solutions, then 5min is cultivated in room temperature darkroom, 5min is exposed afterwards under 500W light, centrifugal, removing supernatant liquor, add DNA extraction liquid again, fully mixing, cultivate, centrifugal, get supernatant liquor as template DNA;
(2) PCR reaction system:
The reaction system of 25 μ L comprises primer and probe described in 18 μ L; The described archaeal dna polymerase of 1 μ L and the mixture of uracil-DNA glycosylase; 1 μ L internal positive control thing; 5 μ L template DNAs or positive control or negative control thing;
(3) PCR response procedures:
4min at 37 DEG C, 95 DEG C of denaturation 5min; 95 DEG C of sex change 10s, 65 DEG C of annealing extend 70s, and 40 circulations, collect FAM, HEX and ROX fluorescent signal simultaneously;
(4) experimental result is judged in conjunction with amplification curve diagram.
Tool of the present invention has the following advantages:
The primer of specific detection enterobacteriaceae lactobacteriaceae of the present invention and/or Enterobacter sakazakii and probe, the characteristic for Enterobacter sakazakii has carried out Idiotype design, can guarantee the accuracy of result.
Test kit of the present invention, the primer utilizing above-mentioned specificity to relate to and probe, and be added with ethidium bromide list nitrine bromine aqueous solution, the cytolemma of dead cell can be penetrated into, covalent linkage is produced with the DNA of killed bacterial, thus prevent it from participating in, in PCR reaction, therefore eliminating the false positive phenomenon produced due to the existence of killed bacterial.
Test kit of the present invention, is added with UNG reagent, can pollute by the residue of degraded PCR primer last time, ensure that the accuracy of result when the first step (37 DEG C of 5min) of PCR reaction.
Test kit of the present invention, is added with internal positive control, can avoid the false negative result occurred due to the existence of PCR inhibitor.If there is PCR inhibitor, so the passage (ROX) of internal positive control and the passage (FAM and HEX) of object bacteria are negative findings, if namely 3 passages are negative findings, result is now null result, should to reform experiment, therefore effectively to avoid the false negative phenomenon occurred due to the existence of PCR inhibitor.
The method of specific detection enterobacteriaceae lactobacteriaceae of the present invention and/or Enterobacter sakazakii, compared with Screening of Media method, the Enterobacter sakazakii in A Product checking sample and enterobacteriaceae required time is used to be less than one day (comprising the time of twice example enrichment).And the whole reagent needed for detection are provided, do not need other extra reagent.All experimental implementation are all carried out in little centrifuge tube, save lab space.
The method of specific detection enterobacteriaceae lactobacteriaceae of the present invention and/or Enterobacter sakazakii, compared with fluorescent PCR method, the Auele Specific Primer that A product comprises and probe (reagent 1) can detect all kinds of Enterobacter sakazakii and enterobacteriaceae through abundant checking, and the bacterial classification for non-Enterobacter sakazakii or enterobacteria is all examined and do not measured.Reagent S can be used for the DNA removing dead cell, avoids false positive results.UNG (reagent 2) can prevent the pollution of residue between different PCR, avoids false positive results equally.Internal positive control (reagent 3) can be monitored in real time to PCR reaction, the false negative result occurred if there is the existence due to PCR inhibitor, so the passage (ROX) of internal positive control is also shown as negative findings, result is now null result, should to reform experiment, therefore effectively to avoid the generation of false negative phenomenon.The negative control of positive control as quality control and test system normal operation is only provided with in commercially available analogous products, do not prevent the internal positive control of false negative result, and A product of the present invention comprises positive control, negative control and internal positive control, experimental result can be judged fast and effectively in conjunction with amplification curve diagram simultaneously.Therefore A product is used to ensure that the validity of result.
Accompanying drawing explanation
Fig. 1 shows the amplification curve of real-time fluorescence quantitative PCR instrument display;
Fig. 2 shows another amplification curve of real-time fluorescence quantitative PCR instrument display.
Embodiment
Hereinafter will be described in detail to embodiments of the invention by reference to the accompanying drawings.It should be noted that, when not conflicting, the embodiment in the application and the feature in embodiment can arbitrary combination mutually.
Embodiment 1
The present embodiment to use in A Product checking food Enterobacter sakazakii in particularly baby formula milk powder and other bacterium of enterobacteriaceae.A product, based on the method for real-time quantitative PCR, comprises following several reagent:
Reagent S: ethidium bromide list nitrine bromine aqueous solution 125 μ g/mL;
Reagent L: be DNA extraction liquid and 0.1%Chelex100 (resin) aqueous solution, can extract the DNA of Gram-negative bacteria (as enterobacteriaceae) in 30min, the DNA extracted can be directly used in the DNA profiling of PCR;
Reagent 1: comprise specific primer and probe, the final concentration of described primer is 0.4 μm of ol/L, and the final concentration of described probe is 0.2 μm of ol/L, MgCl
2, dNTPs, PCR reaction buffer;
The sequential structure of described primer is as follows:
Enterobacteriaceae-F:5 '-CTG AAA GCA TCT AAG CAC GAA ACT TG-3 ';
Enterobacteriaceae-R:5 '-GTC GTC TTC AAC GTT CCT TCA GG-3 ';
Internal positive control-F:5 '-TGT GAA ATA CCG CAC AGA TG-3 ';
Internal positive control (IAC)-R:5 '-AGC TGG CGT AAT AGC GAA G-3 ';
The sequential structure of described probe is as follows:
Enterobacteriaceae lactobacteriaceae probe:
5′-HEX-AGAGTAGTAGTTGTAGAGGCCGTGCTTCCGAAAG-BHQ-1-3′；
Internal positive control probe:
5′-ROX-TAAGGAGAAAATACCGCATCAGGCGCC-BHQ-2-3′；
The enzyme mixture of reagent 2:Taq archaeal dna polymerase and uracil-N-glycosylase;
Reagent 3 is internal positive control, comprises one section of stable plasmid DNA (pUC19) plasmid and strengthens visual yellow dyes;
Reagent 4 is positive control: Enterobacter sakazakii DNA;
Reagent 5 is negative control: PCR level water.
In order to the false positive results preventing dead cell or dissociative DNA from causing, in this test kit, add a kind of sensitivity of light material S.Because this material only can penetrate the cytolemma of killed bacterial, after exposure under visible light, just can there is covalent attachment with DNA in this material, prevents the DNA of dead bacterium from being increased by PCR circulation, avoid the generation of false positive results.Reagent S is developed the dead enterobacteria for removing in instant formula milk.。
The primer that this product test kit provides and probe can the specific DNA in conjunction with enterobacteria or Enterobacter sakazakii, and increase.Supporting quantitative PCR apparatus can carry out synchronous detection to amplification.Probe can with internal reference specific binding, detect in passage ROX, and the DNA of Enterobacter sakazakii (Cronobacter Pseudomonas) is detected at FAM passage, the DNA of enterobacteriaceae is detected at HEX passage.Uracil dna glycosylase (UNG) can prevent the pollution of residue between different PCR.
The method of specific detection enterobacteriaceae lactobacteriaceae described in the present embodiment, specifically comprises:
(1) extraction (using reagent S and reagent L) of DNA
Get sample 100g to be dissolved in and to be preheated to 37 DEG C and to be equipped with in the Erlenmeyer flask of 900mL buffered peptone water, cultivate 18h at 37 DEG C, carry out enrichment first.Draw 450 μ l buffered peptone waters in the centrifuge tube of 1.5ml.Add the sample of 50 μ l enrichments first, mixing.Cultivate 3-4h for 37 DEG C and carry out secondary enrichment.
Draw 300 μ l reagent S in new 1.5ml centrifuge tube, then add the sample of 100 μ l, bis-enrichments, then cultivate 5min in darkroom, under 500W light, expose 5min afterwards.The centrifugal 5min of 8000g, removing supernatant liquor.
Add 200 μ l reagent L, up and down inhale beat or vortex shake make throw out Eddy diffusion.10min is cultivated at using metal heater 95-100 DEG C.Room temperature places 1min afterwards, whirlpool concussion 2s.The centrifugal 2min of 13000g.Supernatant liquor can be directly used in PCR.
(2) PCR reaction system
The reaction system of 25 μ L comprises:
18 μ L reagent 1 (2 pairs of primers (final concentration is 0.4 μm of ol/L), 2 kinds of probes (final concentration is 0.2 μm of ol/L), MgCl
2, dNTPs, PCR reaction buffer);
1 μ L reagent 2 (enzyme mixture of archaeal dna polymerase and uracil-DNA glycosylase);
1 μ L reagent 3 (pUC19 plasmid);
5 μ L template DNAs.
(3) PCR programming: 37 DEG C of 4min, 95 DEG C of denaturation 5min; 95 DEG C of sex change 10s, 65 DEG C of annealing extend 70s, and 40 circulations, collect FAM, HEX and ROX fluorescent signal simultaneously.
(4) experimental result is judged in conjunction with amplification curve diagram
When detecting sample Ct value and being less than or equal to 35, be reported as the positive; When Ct value is greater than 35 and is less than 40, need to repeat once, if Ct value is still less than 40, and curve has obvious increased logarithmic phase, is reported as the positive, otherwise is negative; When sample does not have a Ct value, be reported as feminine gender.Concrete reaction and result judge with reference to table 1.
Table 1 detected result interpretation table
When real-time fluorescence quantitative PCR instrument display amplification curve as shown in Figure 1 time, be judged to be the positive.
When the amplification curve of real-time fluorescence quantitative PCR instrument display is illustrated in fig. 2 shown below, be judged to be feminine gender.
With the test kit described in the present embodiment, qualitative detection is carried out to enterobacteriaceae lactobacteriaceae, detect sample and comprise: infant formula powder (numbering 1-5), sample concentration is 100g/L; With the addition of the powdered milk sample (numbering 6-20) of the rugged bacterium liquid of slope, salmonella liquid, colibacillus liquid respectively; With the addition of the rugged bacterium liquid of slope, salmonella liquid, colibacillus liquid but carried out the powdered milk sample (numbering 21-35) of inactivation treatment respectively; Suction cleaner resistates (numbering 36-40); The cotton swab (numbering 41-45) of wiping device; Positive control (plasmid containing enterobacteriaceae and Enterobacter sakazakii conserved sequence) and negative control (PCR level water) are set simultaneously.The concentration that each sample adds bacterium liquid sees the following form 2.Carry out the contrast (see GB4789.38-2012, GB 4789.4-2010, GB 4789.40-2010) of detected result with existing National Standard Method simultaneously.Wherein, the amplification curve of the PCR instrument display that sample 6 detects is same as shown in Figure 1, is positive findings.
Table 2 enterobacteriaceae lactobacteriaceae detected result
Visible, the detection method of test kit described in the application can comparatively sensitive while detect multiple enterobacteriaceae lactobacteriaceae.
Embodiment 2
The present embodiment to use in A Product checking food Enterobacter sakazakii in particularly baby formula milk powder and other bacterium of enterobacteriaceae.A product, based on the method for real-time quantitative PCR, comprises following several reagent:
Reagent S: ethidium bromide list nitrine bromine aqueous solution 125 μ g/mL;
Reagent L: be DNA extraction liquid and 0.1%Chelex100 (resin) aqueous solution, can extract the DNA of Gram-negative bacteria (as enterobacteriaceae) in 30min, the DNA extracted can be directly used in the DNA profiling of PCR;
Reagent 1: comprise specific primer and probe, the final concentration of described primer is 0.4 μm of ol/L, and the final concentration of described probe is 0.2 μm of ol/L, MgCl
2, dNTPs, PCR reaction buffer;
The sequential structure of described primer is as follows:
Enterobacter sakazakii-F:5 '-AGG GGA TAT TGT CCC CTG AAA CAG-3 ';
Enterobacter sakazakii-R:5 '-AAA CGA GAA TAA GCC GCG CAT T-3 ';
Enterobacteriaceae-F:5 '-CTG AAA GCA TCT AAG CAC GAA ACT TG-3 ';
Enterobacteriaceae-R:5 '-GTC GTC TTC AAC GTT CCT TCA GG-3 ';
Internal positive control-F:5 '-TGT GAA ATA CCG CAC AGA TG-3 ';
Internal positive control (IAC)-R:5 '-AGC TGG CGT AAT AGC GAA G-3 ';
The sequential structure of described probe is as follows:
Enterobacter sakazakii probe:
5′-HEX-AGAGTAGTAGTTGTAGAGGCCGTGCTTCCGAAAG-BHQ-1-3′；
Enterobacteriaceae lactobacteriaceae probe:
5′-HEX-AGAGTAGTAGTTGTAGAGGCCGTGCTTCCGAAAG-BHQ-1-3′；
Internal positive control probe:
5′-ROX-TAAGGAGAAAATACCGCATCAGGCGCC-BHQ-2-3′；
The enzyme mixture of reagent 2:Taq archaeal dna polymerase and uracil-N-glycosylase;
Reagent 3 is internal positive control, comprises one section of stable plasmid DNA (pUC19) plasmid and strengthens visual yellow dyes;
Reagent 4 is positive control: Enterobacter sakazakii DNA;
Reagent 5 is negative control: PCR level water.
In order to the false positive results preventing dead cell or dissociative DNA from causing, in this test kit, add a kind of sensitivity of light material S.Because this material only can penetrate the cytolemma of killed bacterial, after exposure under visible light, just can there is covalent attachment with DNA in this material, prevents the DNA of dead bacterium from being increased by PCR circulation, avoid the generation of false positive results.Reagent S is developed the dead enterobacteria for removing in instant formula milk.
The primer that this product test kit provides and probe can the specific DNA in conjunction with enterobacteria or Enterobacter sakazakii, and increase.Supporting quantitative PCR apparatus can carry out synchronous detection to amplification.Probe can with internal reference specific binding, detect in passage ROX, and the DNA of Enterobacter sakazakii (Cronobacter Pseudomonas) is detected at FAM passage, the DNA of enterobacteriaceae is detected at HEX passage.Uracil dna glycosylase (UNG) can prevent the pollution of residue between different PCR.
The method of specific detection enterobacteriaceae lactobacteriaceae and Enterobacter sakazakii while of described in the present embodiment, specifically comprises:
(1) extraction (using reagent S and reagent L) of DNA
Get sample 100g to be dissolved in and to be preheated to 37 DEG C and to be equipped with in the Erlenmeyer flask of 900mL buffered peptone water, cultivate 18h at 37 DEG C, carry out enrichment first.Draw 450 μ l buffered peptone waters in the centrifuge tube of 1.5ml.Add the sample of 50 μ l enrichments first, mixing.Cultivate 3-4h for 37 DEG C and carry out secondary enrichment.
Draw 300 μ l reagent S (ethidium bromide list nitrine bromine (EMA): 125 μ g/mL) in new 1.5ml centrifuge tube, then add the sample of 100 μ l, bis-enrichments, then cultivate 5min in darkroom, under 500W light, expose 5min afterwards.The centrifugal 5min of 8,000g, removing supernatant liquor.
Then add 200 μ l reagent L (0.1%Chelex100 (resin) aqueous solution), up and down inhale beat or vortex shake make throw out Eddy diffusion.Min is cultivated at using metal heater 95-100 DEG C.Room temperature places 1min afterwards, whirlpool concussion 2s.The centrifugal 2min of 13,000g.Supernatant liquor can be directly used in PCR reaction.
(2) PCR reaction system
The reaction system of 25 μ L comprises:
18 μ L reagent 1 (3 pairs of primers (final concentration is 0.4 μm of ol/L), 3 kinds of probes (final concentration is 0.2 μm of ol/L), MgCl2, dNTPs, PCR reaction buffer);
1 μ L reagent 2 (enzyme mixture of archaeal dna polymerase and uracil-DNA glycosylase);
1 μ L reagent 3 (pUC19 plasmid);
5 μ L template DNAs.
(3) PCR programming: 37 DEG C of 4min, 95 DEG C of denaturation 5min; 95 DEG C of sex change 10s, 65 DEG C of annealing extend 70s, and 40 circulations, collect FAM, HEX and ROX fluorescent signal simultaneously.
(4) experimental result is judged in conjunction with amplification curve diagram.
When detecting sample Ct value and being less than or equal to 35, be reported as the positive; When Ct value is greater than 35 and is less than 40, need to repeat once, if Ct value is still less than 40, and curve has obvious increased logarithmic phase, is reported as the positive, otherwise is negative; When sample does not have a Ct value, be reported as feminine gender.Concrete reaction and result judge with reference to table 3.
Table 3 detected result interpretation table
When real-time fluorescence quantitative PCR instrument display amplification curve as shown in Figure 1 time, be judged to be the positive.
When the amplification curve of real-time fluorescence quantitative PCR instrument display is illustrated in fig. 2 shown below, be judged to be feminine gender.
With the test kit described in the present embodiment, qualitative detection is carried out to enterobacteriaceae lactobacteriaceae, detect sample and comprise: infant formula powder (numbering S1-5), sample concentration is 100g/L; With the addition of the powdered milk sample (numbering S6-20) of the rugged bacterium liquid of slope, salmonella liquid, colibacillus liquid respectively; With the addition of the rugged bacterium liquid of slope, salmonella liquid, colibacillus liquid but carried out the powdered milk sample (numbering S21-35) of inactivation treatment respectively; Suction cleaner resistates (numbering S36-40); The cotton swab (numbering S41-45) of wiping device; Positive control (plasmid containing enterobacteriaceae and Enterobacter sakazakii conserved sequence) and negative control (PCR level water) are set simultaneously.The concentration that each sample adds bacterium liquid sees the following form 4, carries out the contrast (see GB 4789.38-2012, GB 4789.4-2010, GB 4789.40-2010) of detected result with existing National Standard Method simultaneously.Wherein, the amplification curve of the PCR instrument display that sample 6 detects is same as shown in Figure 1, is positive findings.
Table 4 enterobacteriaceae lactobacteriaceae detected result
Visible, the detection method of test kit described in the application can comparatively sensitive while detect multiple enterobacteriaceae lactobacteriaceae, the detection simultaneously for Enterobacter sakazakii also has good detection perform.
Embodiment 3
The method of specific detection enterobacteriaceae lactobacteriaceae and Enterobacter sakazakii while of described in the present embodiment, adopts the test kit described in embodiment 2, specifically comprises:
(1) extraction (using reagent S and reagent L) of DNA
Get sample 100g (mL) to be dissolved in and to be preheated to 37 DEG C and to be equipped with in the Erlenmeyer flask of 900mL buffered peptone water, cultivate 18h at 37 DEG C, carry out enrichment first.Draw 450 μ l buffered peptone waters in the centrifuge tube of 1.5ml.Add the sample of 50 μ l enrichments first, mixing.Cultivate 3-4h for 37 DEG C and carry out secondary enrichment.
Draw 300 μ l reagent S (ethidium bromide list nitrine bromine (EMA): 125 μ g/mL) in new 1.5ml centrifuge tube, then add the sample of 100 μ l, bis-enrichments, then cultivate 5min in darkroom, under 500W light, expose 5min afterwards.The centrifugal 5min of 8,000g, removing supernatant liquor.
Then add 200 μ l reagent L (0.1%Chelex100 (resin) aqueous solution), up and down inhale beat or vortex shake make throw out Eddy diffusion.10min is cultivated at using metal heater 95-100 DEG C.Room temperature places 1min afterwards, whirlpool concussion 2s.The centrifugal 2min of 13,000g.Supernatant liquor can be directly used in PCR reaction.
(2) PCR reaction system
The reaction system of 25 μ L comprises:
18 μ L reagent 1 (3 pairs of primers (final concentration is 0.4 μm of ol/L), 3 kinds of probes (final concentration is 0.2 μm of ol/L), MgCl2, dNTPs, PCR reaction buffer);
1 μ L reagent 2 (enzyme mixture of archaeal dna polymerase and uracil-DNA glycosylase);
1 μ L reagent 3 (pUC19 plasmid);
5 μ L positive controls (Enterobacter sakazakii DNA).
(3) PCR programming: 37 DEG C of 4min, 95 DEG C of denaturation 5min; 95 DEG C of sex change 10s, 65 DEG C of annealing extend 70s, and 40 circulations, collect FAM, HEX and ROX fluorescent signal simultaneously.
(4) experimental result is judged in conjunction with amplification curve diagram,
Embodiment 4
The method of specific detection enterobacteriaceae lactobacteriaceae and Enterobacter sakazakii while of described in the present embodiment, adopts the test kit described in embodiment 2, specifically comprises:
(1) extraction (using reagent S and reagent L) of DNA
Get sample 100g (mL) to be dissolved in and to be preheated to 37 DEG C and to be equipped with in the Erlenmeyer flask of 900mL buffered peptone water, cultivate 18h at 37 DEG C, carry out enrichment first.Draw 450 μ l buffered peptone waters in the centrifuge tube of 1.5ml.Add the sample of 50 μ l enrichments first, mixing.Cultivate 3-4h for 37 DEG C and carry out secondary enrichment.
Draw 300 μ l reagent S (ethidium bromide list nitrine bromine (EMA): 125 μ g/mL) in new 1.5ml centrifuge tube, then add the sample of 100 μ l, bis-enrichments, then cultivate 5min in darkroom, under 500W light, expose 5min afterwards.The centrifugal 5min of 8,000g, removing supernatant liquor.
Then add 200 μ l reagent L (0.1%Chelex100 (resin) aqueous solution), up and down inhale beat or vortex shake make throw out Eddy diffusion.10min is cultivated at using metal heater 95-100 DEG C.Room temperature places 1min afterwards, whirlpool concussion 2s.The centrifugal 2min of 13,000g.Supernatant liquor can be directly used in PCR reaction.
(2) PCR reaction system
The reaction system of 25 μ L comprises:
18 μ L reagent 1 (3 pairs of primers (final concentration is 0.4 μm of ol/L), 3 kinds of probes (final concentration is 0.2 μm of ol/L), MgCl2, dNTPs, PCR reaction buffer);
1 μ L reagent 2 (enzyme mixture of archaeal dna polymerase and uracil-DNA glycosylase);
1 μ L reagent 3 (pUC19 plasmid);
5 μ L negative control things.(PCR level water)
(3) PCR programming: 37 DEG C of 4min, 95 DEG C of denaturation 5min; 95 DEG C of sex change 10s, 65 DEG C of annealing extend 70s, and 40 circulations, collect FAM, HEX and ROX fluorescent signal simultaneously.
(4) experimental result is judged in conjunction with amplification curve diagram.
Due to the employing reagent S of products innovation, effectively can prevent the interference of killed bacterial, thus avoid the generation of false positive results.Use specific probe and primer in conjunction with the DNA conserved sequence of Enterobacter sakazakii and enterobacteriaceae, and Real-Time Monitoring can be carried out in the passage of different wave length.FAM Air conduct measurement Enterobacter sakazakii, HEX Air conduct measurement enterobacteriaceae, ROX Air conduct measurement internal positive control.Namely once experiment can report two results, Enterobacter sakazakii and enterobacteriaceaes simultaneously.In addition, internal positive control, positive control, negative control can guarantee the accuracy of experimental result jointly.
Although the embodiment disclosed by the present invention is as above, the embodiment that described content just adopts for the ease of understanding the present invention, and be not used to limit the present invention.Technician in any the technical field of the invention; under the prerequisite not departing from the spirit and scope disclosed by the present invention; any amendment and change can be done what implement in form and in details; but scope of patent protection of the present invention, the scope that still must define with appending claims is as the criterion.
Claims (10)
1. a primer for specific detection enterobacteriaceae lactobacteriaceae, is characterized in that, the sequential structure of described primer is as follows:
Enterobacteriaceae-F:5 '-CTG AAA GCA TCT AAG CAC GAA ACT TG-3 ';
Enterobacteriaceae-R:5 '-GTC GTC TTC AAC GTT CCT TCA GG-3 ';
Internal positive control-F:5 '-TGT GAA ATA CCG CAC AGA TG-3 ';
Internal positive control (IAC)-R:5 '-AGC TGG CGT AAT AGC GAA G-3 '.
2. for a probe for specific detection Enterobacter sakazakii, it is characterized in that, the sequential structure of described probe is as follows:
Enterobacteriaceae lactobacteriaceae probe:
5 '-HEX-AGAGTAGTAGTTGTAGAGGCCGTGCTTCCGAAAG-BHQ-1-3 '; Internal positive control probe:
5′-ROX-TAAGGAGAAAATACCGCATCAGGCGCC-BHQ-2-3′。
3. for a test kit for specific detection enterobacteriaceae lactobacteriaceae, it is characterized in that: comprise primer according to claim 11 and probe according to claim 12, and sensitivity of light material.
4. the test kit for specific detection enterobacteriaceae lactobacteriaceae according to claim 3, it is characterized in that: described sensitivity of light material comprises and can form ethidium bromide list nitrine bromine (EMA) of covalent linkage with the base of DNA, and its concentration is 125 μ g/mL.
5. the test kit for specific detection enterobacteriaceae lactobacteriaceae according to claim 3 or 4, is characterized in that, described test kit also comprises:
Internal positive control thing, described internal positive control thing comprises pUC19 plasmid DNA;
The mixture of archaeal dna polymerase and uracil-DNA glycosylase;
The DNA of enterobacteriaceae lactobacteriaceae is positive control, the contrast of PCR level water belongs with yin;
Buffered peptone water and DNA extraction liquid;
And optionally add visual yellow dyes.
6. a method for specific detection enterobacteriaceae lactobacteriaceae, is characterized in that, adopt as the test kit in claim 3-5 as described in any one, this detection method comprises the following steps:
(1) extraction of DNA:
Get measuring samples and carry out multiplication culture, enrichment, 300 μ l ethidium bromide list nitrine bromines are added in 100 μ l pregnant solutions, then 5min is cultivated in room temperature darkroom, 5min is exposed afterwards under 500W light, centrifugal, removing supernatant liquor, add DNA extraction liquid again, fully mixing, cultivate, centrifugal, get supernatant liquor as template DNA;
(2) PCR reaction system:
The reaction system of 25 μ L comprises primer and probe described in 18 μ L; The described archaeal dna polymerase of 1 μ L and the mixture of uracil-DNA glycosylase; 1 μ L internal positive control thing; 5 μ L template DNAs or positive control or negative control thing;
(3) PCR response procedures:
4min at 37 DEG C, 95 DEG C of denaturation 5min; 95 DEG C of sex change 10s, 65 DEG C of annealing extend 70s, and 40 circulations, collect FAM, HEX and ROX fluorescent signal simultaneously;
(4) experimental result is judged in conjunction with amplification curve diagram.
7., for a test kit for specific detection enterobacteriaceae and Enterobacter sakazakii, it is characterized in that: comprising:
Enterobacter sakazakii-F:5 '-AGG GGA TAT TGT CCC CTG AAA CAG-3 ';
Enterobacter sakazakii-R:5 '-AAA CGA GAA TAA GCC GCG CAT T-3 ';
Enterobacteriaceae-F:5 '-CTG AAA GCA TCT AAG CAC GAA ACT TG-3 ';
Enterobacteriaceae-R:5 '-GTC GTC TTC AAC GTT CCT TCA GG-3 ';
Internal positive control-F:5 '-TGT GAA ATA CCG CAC AGA TG-3 ';
Internal positive control (IAC)-R:5 '-AGC TGG CGT AAT AGC GAA G-3 ';
Enterobacter sakazakii probe:
5′-HEX-AGAGTAGTAGTTGTAGAGGCCGTGCTTCCGAAAG-BHQ-1-3′；
Enterobacteriaceae probe:
5′-HEX-AGAGTAGTAGTTGTAGAGGCCGTGCTTCCGAAAG-BHQ-1-3′；
Internal positive control probe:
5′-ROX-TAAGGAGAAAATACCGCATCAGGCGCC-BHQ-2-3′；
And sensitivity of light material.
8. the test kit for specific detection enterobacteriaceae and Enterobacter sakazakii according to claim 7, it is characterized in that: described sensitivity of light material comprises and can form ethidium bromide list nitrine bromine (EMA) of covalent linkage with the base of DNA, and its concentration is 125 μ g/mL.
9. the test kit according to claim 7 or 8, is characterized in that, described test kit also comprises:
Internal positive control thing, described internal positive control thing comprises pUC19 plasmid DNA;
The mixture of archaeal dna polymerase and uracil-DNA glycosylase;
The DNA of enterobacteriaceae lactobacteriaceae is positive control, the contrast of PCR level water belongs with yin;
Buffered peptone water and DNA extraction liquid;
And optionally add visual yellow dyes.
10. a method for specific detection enterobacteriaceae lactobacteriaceae and Enterobacter sakazakii, is characterized in that, adopt as the test kit in claim 7-9 as described in any one, this detection method comprises the following steps:
(1) extraction of DNA:
Get measuring samples and carry out multiplication culture, enrichment, 300 μ l ethidium bromide list nitrine bromines are added in 100 μ l pregnant solutions, then 5min is cultivated in room temperature darkroom, 5min is exposed afterwards under 500W light, centrifugal, removing supernatant liquor, add DNA extraction liquid again, fully mixing, cultivate, centrifugal, get supernatant liquor as template DNA;
(2) PCR reaction system:
The reaction system of 25 μ L comprises primer and probe described in 18 μ L; The described archaeal dna polymerase of 1 μ L and the mixture of uracil-DNA glycosylase; 1 μ L internal positive control thing; 5 μ L template DNAs or positive control or negative control thing;
(3) PCR response procedures:
4min at 37 DEG C, 95 DEG C of denaturation 5min; 95 DEG C of sex change 10s, 65 DEG C of annealing extend 70s, and 40 circulations, collect FAM, HEX and ROX fluorescent signal simultaneously;
(4) experimental result is judged in conjunction with amplification curve diagram.
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Application publication date: 20151028 |